L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100

ABSTRACT

A novel L-sorbose dehydrogenase (SDH) and a novel L-sorbosone dehydrogenase both derived from Gluconobacter oxydans T-100, a DNA which encodes the SDH and/or SNDH, an expression vector which contains the DNA, a host cell transformed by the expression vector and a process for producing the SDH and/or SNDH, which comprises culturing the host cell in a medium and recovering the SDH and/or SNDH from the resulting culture. The SDH and SNDH of the present invention are useful enzymes having preferable properties for the production of 2-keto-L-gulonic acid, as well as L-ascorbic acid. According to the production method of the present invention, the SDH and SNDH having such preferable properties can be produced in large amounts by genetic engineering.

This application is a Continuation of application Ser. No. 08/942,673,filed on Oct. 2, 1997, now U.S. Pat. No. 5,861,292, which is aContinuation of application Ser. No. 08/513,841, filed on Nov. 1, 1995,now U.S. Pat. No. 5,753,481, which was filed as InternationalApplication No. PCT/JP94/00369, filed on Mar. 8, 1994.

TECHNICAL FIELD

The present invention relates to a novel L-sorbose dehydrogenase(hereinafter referred to as SDH) and a novel L-sorbosone dehydrogenase(hereinafter referred to as SNDH) both derived from GluconobacterOxydans T-100. More particularly, the present invention relates to anovel SDH, a novel SNDH, a DNA encoding same, an expression vectorcontaining said DNA, a host cell transformed (transfected) with saidexpression vector, and the production of the SDH and SNDH by culturingthe host cell.

The SDH and SNDH of the present invention are enzymes useful forproducing 2-keto-L-gulonic acid.

BACKGROUND ART

2-Keto-L-gulonic acid (hereinafter referred to as 2KLGA) is a keyintermediate in the synthesis of L-ascorbic acid. For industrialproduction, 2KLGA is chemically synthesized from L-sorbose by oxidationaccording to the Reichstein's method.

On the other hand, it is well known that many microorganisms have anability to convert L-sorbose to 2KLGA through a two-step enzymaticoxidation by SDH and SNDH. Namely, SDH catalyzes the. oxidation ofL-sorbose to L-sorbosone, and SNDH catalyzes the oxidation ofL-sorbosone to 2KLGA. However, because of the low productivity of 2KLGAobtained by using these microorganisms, they have not been applied tothe industrial production yet.

It is desirable to provide efficient and simplified methods for theproduction of 2KLGA.

DISCLOSURE OF THE INVENTION

In an attempt to accomplish the above-mentioned objects, the inventorsof this invention conducted intensive studies to find an SDH and an SNDHhaving preferable properties, and succeeded in producing a novel SDH anda novel SNDH having desirable properties for producing 2KLGA anddeveloped the studies, which resulted in the completion of the presentinvention.

Accordingly, the present invention relates to a novel SDH derived fromGluconobacter oxydans T-100 (FERM BP-4188), which is characterized by:

(1) an ability to catalyze the conversion of L-sorbose into L-sorbosone,

(2) a molecular weight of 58,000 dalton (SDS-PAGE), and

(3) an N-terminal amino acid sequence ofThr—Ser—Gly—Phe—Asp—Tyr—Ile—Val—Val—Gly—Gly—Gly—Ser—Ala(SEQ ID NO: 5).Further, the present invention relates to an SDH having an amino acidsequence shown in the Sequence Listing, Sequence No. 1 to be mentionedlater.

The present invention also relates to a novel SNDH derived fromGluconobacter oxydans T-100, which is characterized by:

(1) an ability to catalyze the conversion of L-sorbosone into 2KLGA,

(2) a molecular weight of 50,000 dalton (SDS-PAGE), and

(3) an N-terminal amino acid sequence ofAsn—Val—Val—Ser—Lys—Thr—Val—Xaa—Leu (SEQ ID NO: 6, Xaa being anunidentified amino acid). The present invention further relates to anSNDH having an amino acid sequence shown in the Sequence Listing,Sequence No. 2.

The present invention also relates to a DNA which encodes theabove-mentioned SDH and/or SNDH, an expression vector which containssaid DNA, a host cell transformed (transfected) by said expressionvector and a process for producing the SDH and/or SNDH, which comprisesculturing said host cell (transformant) in a medium and recovering theSDH and/or SNDH from the resulting culture.

The Gluconobacter oxydans T-100 to be used in the present invention is a2KLGA-high-producing mutant derived from Gluconobacter oxydans G716(wild strain) by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenesisin a conventional manner.

The Gluconobacter oxydans G716 was isolated from a persimmon, and hasthe following morphological and physiological properties. The methoddescribed in Bergey's Manual of Systematic Bacteriology Vol. 1 (1984)and the method described in Manual for Identification to MedicalBacteria (S. T. Cowan, 2nd. Edition, 1985) were principally employed forthe taxonomic study.

1. Morphological properties

The Gluconobacter oxydans G716 is a Gram-negative, motile bacterium. Thecell shapes are rod, occurring both singly and in pairs, and rarely inchains. Morphological characteristics of Gluconobacter oxydans G716

Gram stain negative color of colony pale spore negative cell shape rodmotility positive flagella 3-8 polar flagella

2. Physiological characteristics

Physiological characteristics of the Gluconobacter oxydans G716 aresummarized in the following table.

Physiological characteristics of Gluconobacter oxydans G716

Conditions Characteristics growth in air + at 4° C. − at 22° C. + at 30°C. + at 40° C. − catalase + oxidase − gelatin liquefaction − nitratereduction − aesuclin hydrolysis − acid formation L-arabinose +D-cellobiose + Dulcitol + D-galactose + D-glucose + glycerol +D-mannitol + D-mannose + D-xylose + D-lactose − maltose − D-raffinose −rhamnose − D-sorbitol − sucrose − D-trehalose − mol% G + C of the DNA60.0 Ubiquinone Q10

The organism is aerobic, showing no growth under anaerobic conditions.Optimum temperature is 22 to 30° C., showing no growth at 40° C. and 4°C. The best medium for growth is SY medium that is composed of 2.5%sorbitol and 0.5% yeast extract (pH 6.4). Strong ketogenesis occurs fromglucose and glycerol.

The Gluconobacter oxydans T-100 to be used in the present invention hasthe morphological and physiological properties identical to those ofGluconobacter oxydans G716.

The new SDH and new SNDH of the present invention can be prepared byrecombinant DNA technology, polypeptide synthesis and the like.

In case where recombinant DNA technology is employed, the new SDH and/ornew SNDH can be prepared by culturing a host cell transformed(transfected) with an expression vector containing a DNA encoding theamino acid sequence of the new SDH and/or new SNDH in a nutrient mediumand recovering the same from the obtained culture.

Particulars of this process are explained in more detail in thefollowing.

The host cell includes, for example, microorganisms such as bacteria(e.g. Escherichia coli, Gluconobacter oxydans and Bacillus subtilis),yeast (e.g. Saccharomyces cerevisiae), animal cell lines and culturedplant cells. Preferred examples of the microorganisms include bacteria,especially strains belonging to the genus Escherichia (e.g. E. coliJM109 ATCC 53323, E. coli NM538 ATCC 35638, E. coli HB101 ATCC 33694, E.coli HB101-16 FERM BP-1872 and E. coli 294 ATCC 31446) and the genusBacillus (e.g. Bacillus subtilis ISW1214), yeast, especially strainsbelonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiaeAH22), and animal cell lines [e.g. mouse L929 cell and Chinese hamsterovary (CHO) cell].

When a bacterium, especially E. coli or Bacillus subtilis is used as ahost cell, expression vector is usually composed of at least promoter,initiation codon, DNA encoding amino acid sequence(s) of the new SDHand/or new SNDH, termination codon, terminator region, and replicatableunit.

When a yeast or an animal cell is used as a host cell, the expressionvector is preferably composed of at least promoter, initiation codon,DNA encoding amino acid sequences of signal peptide and the new SDHand/or new SNDH and termination codon, and it is possible that enhancersequence, 5′- and 3′-noncoding region of the new SDH, 5′- and3′-noncoding region of the new SNDH, splicing junctions, polyadenylationsite and replicatable unit are also inserted.

The promoter for expressing the new SDH and/or new SNDH in bacteriacomprises, for example, promoter and Shine-Dalgarno (SD) sequence (e.g.AAGG). Preferable promoters include, for example, conventionallyemployed promoters (e.g. PL-promoter and trp-promoter for E. coli) andpromoter of the SNDH chromosomal gene.

The promoters for expressing the new SDH and/or new SNDH in yeastinclude, for example, the promoter of the TRP1 gene, the ADHI or ADHIIgene, and acid phosphatase (PH05) gene for S. cerevisiae.

The promoters for expressing the new SDH and/or new SNDH in mammaliancells include, for example, SV40 early or late-promoter,HTLV-LTR-promoter, mouse metallothionein I (MMT)-promoter andvaccinia-promoter.

Preferable initiation codon includes, for example, methionine codon(ATG).

The signal peptide includes, for example, signal peptides of otherenzymes conventionally employed (e.g. signal peptide of the native t-PAand signal peptide of the native plasminogen).

The DNA encoding the signal peptide or the new SDH and/or new SNDH canbe prepared in a conventional manner, such as a partial or whole DNAsynthesis using DNA synthesizer and a method comprising preparing fromGluconobacter oxydans genomic DNA in a conventional manner such as PCRprocedure or DNA probe procedure described in Molecular Cloning (mainlyin Chapters 11 and 14, Cold Spring Harbor Laboratory Press, 1989, USA).

The termination codon includes, for example, conventionally employedtermination codons (e.g. TAG and TGA).

The terminator region includes, for example, natural or syntheticterminator (e.g. terminator of the new SDH chromosomal gene andsynthetic fd phage terminator).

The replicatable unit is a DNA compound capable of replicating the wholeDNA sequence belonging thereto in host cell, and may include naturalplasmid, artificially modified plasmid (e.g. DNA fragment prepared fromnatural plasmid) and synthetic plasmid. In the present invention, thereplicatable unit can be appropriately selected according to themicroorganism to be used as a host cell. Preferable examples of theplasmid include plasmid pBR322 and artificially modified plasmid thereof(DNA fragment obtained from a suitable restriction enzyme treatment ofpBR322) for E. coli, yeast 2 μ plasmid and yeast chromosomal DNA foryeast, plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmidpdBPV-MMTneo ATCC 37224 and plasmid pSV2neo ATCC 37149 for mammaliancells.

The enhancer sequence includes, for example, the enhancer sequence (72b.p.) of SV40.

The polyadenylation site includes, for example, the polyadenylation siteof SV40.

The splicing junction includes, for example, the splicing junction ofSV40.

The promoter, initiation codon, DNA encoding amino acid sequence of thenew SDH and/or new SNDH, termination codon(s) and terminator region canconsecutively and circularly be linked with an adequate replicatableunit (plasmid), using, if desired, adequate DNA fragment(s) in aconventional manner (e.g. digestion with restriction enzyme, ligationusing T4 DNA ligase) to give the expression vector of the presentinvention.

When mammalian cells are used as host cells, it is possible thatenhancer sequence, promoter, 5′-noncoding region of the cDNA of the newSDH and/or new SNDH, initiation codon, DNA encoding the signal peptide,DNA encoding amino acid sequence(s) of the new SDH and/or new SNDH,termination codon(s), 3′-noncoding region of the cDNA of the new SDHand/or new SNDH, splicing junctions and polyadenylation site areconsecutively and circularly linked with an adequate replicatable unitin the above manner to give an expression vector.

The transformant of the present invention can be prepared by introducingthe expression vector obtained above into a host cell. Introduction ofthe expression vector into the host cell (transformation, hereinafterused as also meaning transfection) can be carried out in a conventionalmanner (e.g. Kushner method for E. coli, calcium phosphate method formammalian cells and microinjection).

For the production of the new SDH and/or new SNDH by the process of thisinvention, the thus-obtained transformant containing the expressionvector is cultured in an aqueous nutrient medium.

The nutrient medium to be used may contain carbon source(s) (e.g.glucose, glycerine, mannitol, fructose and lactose) and inorganic ororganic nitrogen source(s) (e.g. ammonium sulfate, ammonium chloride,hydrolysate of casein, yeast extract, polypeptone, Bactotrypton and beefextract). If desired, other nutritious sources such as inorganic salts(e.g. sodium or potassium biphosphate, dipotassium hydrogen phosphate,magnesium chloride, magnesium sulfate and calcium chloride), vitamins(e.g. vitamin B₁), and antibiotics (e.g. ampicillin, kanamycin) may beadded to the medium. For the culture of mammalian cells, Dulbecco'sModified Eagle's Minimum Essential Medium (DMEM) supplemented with fetalcalf serum and antibiotic is often used.

The culture of the tranformant is usually carried out at pH 5.5-8.5(preferably pH 7-7.5) and 18-40° C. (preferably 20-30° C.) for 5-50hours.

When the thus-produced new SDH and/or new SNDH exist(s) in the culturesolution, culture filtrate (supernatant) is obtained by filtration orcentrifugation of the culture. The new SDH and/or new SNDH can bepurified from the culture filtrate by a method generally employed forthe purification and isolation of natural or synthetic proteins (e.g.dialysis, gel filtration, affinity column chromatography using anti-SDHmonoclonal antibody or anti-SNDH monoclonal antibody, columnchromatography on a suitable adsorbent and high performance liquidchromatography).

When the produced new SDH and/or new SNDH exist(s) in periplasm andcytoplasm of the cultured transformant, cells are collected byfiltration and centrifugation, and the cell wall and/or cell membranethereof are/is destroyed by, for example, treatment with supersonicwaves and/or lysozyme to give debris. The debris can be dissolved in asuitable aqueous solution (e.g. 8 M aqueous urea and 6 M aqueousguanidium salt). From the solution, the new SDH and/or new SNDH can bepurified in a conventional manner as exemplified above.

If it is necessary to refold the new SDH and/or new SNDH produced in E.coli by the method of the present invention, the refolding can becarried out in a conventional manner.

If the SDH activity and/or new SNDH activity exist(s) in thetransformant, the following can be exemplified as the materials obtainedby processing the culture (hereinafter the material is referred to asprocessed material).

(1) Raw cells: separated from the culture in a conventional manner suchas filtration and centrifugation:

(2) dried cells: obtained by drying said raw cells of (1) above in aconventional manner such as lyophilization and vacuum drying;

(3) cell-free extract: obtained by destroying said raw cells of (1)above or dried cells of (2) above in a conventional manner (e.g.autolysis of the cells using an organic solvent, grinding the cells withalumina, sea sand etc., and treating the cells with supersonic waves);

(4) enzyme solution: obtained by purification or partial purification ofsaid cell-free extracts of (3) above in a conventional manner (e.g.column chromatography); and

(5) immobilized cells or enzyme: prepared by immobilizing said raw cellsof (1) above or dried cells of (2) above or the enzyme of (4) above in aconventional manner (e.g. a method using acrylamide, glass bead, ionexchange resin etc.).

If the SDH activity and/or new SNDH activity exist(s) in a culturefiltrate of the transformant, the culture filtrate (supernatant),purified enzyme solution and immobilized enzymes can be exemplified asthe processed materials of the culture.

The assay of the new SDH activity in crude mixture such as sonicatedcell lysate or its processed material obtained in each purification stepcan be usually conducted according to T. SUGISAWA et al. (Agric. Biol.Chem., 55, 363-370, 1991) using L-sorbose as a substrate and2,6-dichlorophenolindophenol (hereinafter referred to as DCIP) as anelectron acceptor in phosphate buffer (pH 7.0). The enzyme activity ismeasured as the reduction rate of DCIP, which is determined by thedecrease of absorbance at 600 nm. One enzyme unit is defined as theamount of the enzyme that catalyzes the reduction of 1 μmol DCIP perminute. Preferable pH of the reaction mixture, concentration ofL-sorbose and DCIP, reaction time and reaction temperature may vary withthe medium or its processed material to be used. Generally, the reactionis carried out at pH 7 to 10, preferably pH 8 to 9, at 5 to 50° C.,preferably 20 to 45° C. for 0.5 to 24 hours.

The new SDH enzyme activity may be also assayed by determining theamount of the reaction product, L-sorbosone labelled with benzamidinehydrochloride, using post column high performance liquid chromatography(HPLC) with fluorescent detection (Ex. 315 nm, Em. 405 nm).

The activity of novel SNDH present in crude mixtures, such asultrasonication cell lysate and treated substances obtained inrespective purification steps, can be also assayed by a general methodby SUGISAWA et al. (Agric. Biol. Chem., 55, 363-370, 1991). In thiscase, SNDH activity is assayed by measuring the amount of NADH producedusing nicotinamido adenine dinucleotide (hereinafter referred to as AND)as an electron acceptor and L-sorbosone as a substrate in phosphatebuffer (pH 7.0), which is determined by the absorbance at 340 nm. Oneenzyme unit is defined as the amount of the enzyme that produces 1 μmolNADH per minute. Preferable pH of the reaction mixture, concentrationsof L-sorbosone and AND to be used, reaction time and reactiontemperature may vary with a medium and processed materials to be used.Generally, the reaction is carried out at pH 7 to 10, preferably pH 8 to9, at 5 to 50° C., preferably 20 to 45° C. for 0.5 to 24 hours.

The new SDH and new SNDH of the present invention are enzymes havingpreferable properties useful for producing 2KLGA, and thereforL-ascorbic acid. According to the present invention, the new SDH and newSNDH having such preferable properties can be produced in large amountsby genetic engineering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction enzyme map of pUC18SD180.

FIG. 2 is a restriction enzyme map of plasmid pUC19SD5 containing DNAencoding the novel SDH of the present invention and DNA encoding thenovel SNDH of the present invention.

FIG. 3 shows the construction of plasmid pUC19SD5.

The present invention is explained in more detail in the following. Inthe following Examples, plasmids, enzymes such as restriction enzyme, T4DNA ligases, and other materials were obtained from commercial sourcesand used according to the indication by suppliers. Operations employedfor the cloning of DNA, transformation of host cells, cultivation oftransformants, recovery of the new SDH and/or new SNDH from the obtainedculture, and the like are well known in the art or can be adapted fromliteratures.

It is needless to say that the present invention is not limited to theseExamples.

EXAMPLE 1 Purification of SDH from Gluconobacter oxydans T-100

(1) Microorganism

Gluconobacter oxydans T-100 was selected as a 2KLGA-high-producingmutant derived from Gluconobacter oxydans G716 (wild strain) bynitrosoguanidine mutagenesis.

(2) Cultivation of Gluconobacter oxydans T-100

Single colonies of Gluconobacter oxydans T-100 were transferred into 6independent culture media (100 ml each) consisting of 2.5% glucose, 1.0%polypeptone, 0.5% yeast extract (Difco Labs., USA) and 2.0% CaCO₃ in 500ml Erlenmeyer flasks. The cultivation was performed at 30° C. on arotary shaker (250 rpm) for 18 hours. The cultivated medium (total 600ml) was inoculated to 20 liters of a fermentation medium containing 5%D-sorbitol, 0.5% glycerol, 0.5% yeast extract and 1.0% CaCO₃ in a 30 Ljar. Cultivation was carried out at 30° C. for 42 hours under aerationat 20 liters/minute and agitation at 300 rpm. The cultivated broth (20L) was centrifuged at 6,000 rpm at 4° C. for 10 minutes. The cells werewashed once with cold saline and recentrifuged under the sameconditions. The cells were stored at −20° C. until use.

(3) Preparation of the membrane fraction

Cells (17.7 g, wet weight) obtained in (2) were suspended in 50 ml of 10mM phosphate buffer (pH 7.0), disrupted by sonication, and centrifugedat 8,000 rpm at 4° C. for 10 min to give a supernatant. On the otherhand, the resulting precipitates were suspended in 40 ml of 10 mMphosphate buffer (pH 7.0), sonicated for disruption and centrifugedunder the same conditions as above. The supernatants were pooled andultracentrifuged at 32,000 rpm at 4° C. for one hour. The resultingprecipitates were washed once with phosphate buffer (50 ml) andsubjected to ultracentrifugation under the same conditions as above togive crude membrane proteins (membrane fraction).

(4) Solubilization of SDH from the membrane fraction

The membrane fraction obtained in (3) was suspended in 50 ml of 10 mMphosphate buffer (pH 7.0). To the suspension, 0.75 ml of 20% TritonX-100 (Nacalai Tesque, Japan) and 1.8 g of L-sorbose were added, and themixture was stirred on ice for 3.5 hours. The resultant suspension wasultracentrifuged at 32,000 rpm at 4° C. for one hour to give asupernatant (ca. 48 ml), designated as solubilized SDH fraction.

(5) Ion-exchange chromatography

The solubilized fraction (16 ml) obtained in (4) was subjected toion-exchange chromatography on a TSKgel DEAE-5PW column (7.5 mm innerdiameter×75 mm, Toso Co. Ltd., Japan) equilibrated with 10 mM phosphatebuffer (pH 7.0) containing 0.3% Triton k-100 and 200 mM L-sorbose. Thecolumn was eluted with a linear gradient of sodium chloride from 0 M to0.5 M in an equilibration buffer. Enzyme activity was assayed accordingto T. SUGISAWA et al. (Agric. Biol. Chem., Vol. 55, 363-370, 1991) usingL-sorbose as a substrate and 0.1 mM 2,6-dichlorophenolindophenol (DCIP)as an electron acceptor in 0.28 M phosphate buffer (pH 7.0). One enzymeunit was defined as the amount of the enzyme that catalyzes thereduction of 1 μmole DCIP per minute. The reduction of DCIP wasdetermined by the decrease of absorbance at 600 nm withspectrophotometer (Model UV-160, Shimadzu, Japan). Active fractions werepooled, diluted 3-fold with 10 mM phosphate buffer (pH 7.0), and appliedto a DEAE-TOYOPEARL 650 M column (7.0 mm inner diameter ×17 mm, Toso Co.Ltd., Japan) equilibrated with 10 mM phosphate buffer (pH 7.0). Thecolumn was eluted with 0.2 M sodium chloride in an equilibration buffer.The resulting eluate was used for further purification steps.

(6) Gel-filtration chromatography

A portion (300 μl) of the concentrated active fraction was subjected togel-filtration chromatography on a Superose 12 HR10/30 column (10 mminner diameter×30 cm, Pharmacia, Sweden) equilibrated with 10 mMphosphate buffer (pH 7.0) containing 0.3% Triton X-100, 200 mM L-sorboseand 0.2 M sodium chloride. Elution was performed using the same buffer.Each fraction (0.4 ml) was analyzed by polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE,12.5% gel) and by enzyme assay described in Example 1 (5). From theanalysis of SDS-PAGE, SDH activity was found to correspond to 58 kdprotein, suggesting that the 58 kd protein was the desired SDH molecule.

EXAMPLE 2 Amino acid sequence analysis of SDH

The concentrated active fraction (15 μl) was subjected to SDS-PAGE(12.5% gel) and the separated proteins were blotted on a polyvinylidenedifluoride (PYDF) membrane. The membrane containing 58 kd proteinstained by ponceau S was cut out and washed with distilled water. Themembrane piece was directly sequenced with an automated proteinsequencer Model 470A (Applied Biosystems Inc., USA) for N-terminal aminoacid sequence analysis.

To determine the internal amino acid sequence, fragmentation withachromobacter protease I (Wako Chemical, Japan) was carried out on thesurface of the membrane. The fragments obtained were eluted with 50 mMTris-HCl (pH 9.0) containing 8% acetonitrile, and separated by reversedphase chromatography using Cosmosil 5C4-300 (4.6 mm inner diameter×50mm, Nacalai Tesque, Japan) with a linear gradient elution (75 min) ofacetonitrile of from 8% to 83% in 0.05% trifluoroacetic acid. Two kindsof peptides (Peptide 1 and Peptide 2) were isolated, and sequenced withan automated protein sequencer Model 470A for amino acid sequenceidentification. The resultant data are shown in Table 1(SEQ ID NO:5,7,8).

TABLE 1 NH₂-terminal sequence TSGFDYIVVGGGSA Peptide 1 MTTGPHTWDLLTEPQKPeptide 2 LMMLSGVGPA

EXAMPLE 3 Preparation of DNA probe

(1) Synthesis of DNA oligomers

Each oligonucleotide listed in Table 2 below was synthesized by phosphoamidite method using DNA synthesizer model 392 (Applied Biosystems Inc.,USA). The synthesized oligonucleotide was liberated from CPG polymersupport (CPG: controlled pore glass) with 28% aqueous ammonia, followedby heating at 60° C. for 9 hours to remove all protective groups. Thereaction mixture was evaporated in vacuo, and the residue was dissolvedin 200 μL of TE [10 mM Tris-HCl (pH 7.4)-1 mM EDTA]. The resultingsolution was washed once with ether and precipitated with ethanol. Theobtained oligonucleotides were used as primers for polymerase chainreaction without further purification (SEQ ID NO: 9,10).

TABLE 2 Oligonucleotide encoding NH₂-terminal sequence (forward primer)  5′> ACC (TA)(GC)C GGC TT(TC) GA(TC) TA(TC) AT(TCA) GT <3′Oligonucleotide encoding internal Sequence (reverse primer)   5′> TC CCA(ATCG)GT (AG)TG (ATCG)GG (ATCG)CC <3′

(2) Preparation of chromosomal DNA

A single colony of Gluconobacter oxydans T-100 was cultivated in amedium (100 ml) consisting of 2% glucose, 1% polypeptone and 0.5% yeastextract at 37° C. for 24 hours. The cells were collected bycentrifugation (4,600 rpm, 10 min) and suspended in TE buffer (2.5 ml).A portion (2.0 ml) of the suspension was diluted with 20 ml of STEbuffer [20% sucrose-50 mM Tris-HCl (pH 8)−1 mM EDTA], mixed with 5 ml oflysozyme solution (5 mg/ml), and incubated at 37° C. for 30 min.Sarcosil solution [1% lauroyl sarcosilate-100 mM EDTA (pH 9.0)] (50 ml)and proteinase K (40 mg) were added, and the mixture was incubated at50° C. for 1.5 hours. Cesium chloride (93.8 g) and 6 ml of ethidiumbromide (5 mg/ml) were dissolved in 75 ml of said mixture, and thecesium chloride solution was ultracentrifuged at 50,000 rpm at 20° C.for 14 hours. The portion containing chromosomal DNA was isolated,washed twice with isopropyl alcohol saturated with physiological saline,and dialyzed against TE buffer (2 L) for 4 hours. The dialysate wasextracted with phenol (20 ml), and dialyzed twice against TE buffer (2L) to give the desired chromosomal DNA solution (14 ml, 91.5 μg/ml).

(3) Polymerase chain reaction

Polymerase chain reaction (PCR) was carried out with 180 ng ofGluconobacter oxydans T-100 chromosomal DNA and 2.5 μmoles of eachprimer of Table 2, using Hybaid thermal reactor Model HB-TR1 (HybaidLimited, UK). The reaction mixture [200 μM dNTPs each and 2.5 units TaqDNA polymerase in PCR buffer (Perkin Elmer-Cetus, USA)] was subjected to50 cycles of PCR, each consisting of 0.5 min of denaturation at 95° C.,1 min of annealing at 42° C. and 2 min of polymerization at 72° C. Asingle fragment was obtained by PCR. The DNA fragment (180 bp)supposedly coding for a part of the SDH gene was isolated by 1.5%agarose gel-electrophoresis and filled with DNA polymerase Klenowfragment (Takara Shuzo, Japan) to give a blunt-ended DNA. The resultantDNA and pUC18 previously digested with SmaI (Nippon Gene, Japan) wereligated in the presence of T4 DNA ligase (Takara Shuzo, Japan). Theligation mixture was used to transform E. coli JM109 (Nippon Gene,Japan) according to the procedure of SHIGESADA et al. (Saibo-kogaku, 2,616-626, 1983). From one of the transformants, the desired plasmidpUC18SD180 (see FIG. 1) was obtained and characterized by restrictionmapping.

(4) Preparation of the ³²P-labelled probe

The insert DNA (ca. 200 bp) was isolated by digestion of pUC18SD180 withBamHI and EcoRI (Nippon Gene, Japan). The ca. 200 bp DNA was purified by0.5% agarose gel-electrophoresis. Purified DNA was ³²P-labelled withnick translation kit (Takara Shuzo, Japan) according to the appendedprotocols. The specific activity of DNA labelled with 32p was about3.7×10⁷ cpm/μg.

EXAMPLE 4 Isolation of SDH gene from Gluconobacter oxydans T-100 DNAlibrary

(1) Preparation of chromosomal DNA library

The genomic DNA obtained in Example 3 (2) was partially digested withMboI (Nippon Gene, Japan) and the fragments were separated on a sucrosegradient to produce a fragment with a size range of from 8 kbp to 22 kbpbefore cloning into the BamHI site of lambda phage vector EMBL-3(Clonetech). This lambda phage vector was introduced into E. coli NM538(Clonetech) to construct Gluconobacter T-100 chromosomal DNA library.

(2) Plaque hybridization

Preparation of lambda phage plaques with E. coli NM538 (Clonetech) as aplating bacterium and immobilization of the plaques on thenitrocellulose filter were carried out according to the protocolsdescribed in Molecular Cloning vol. 1, Chapter 2, page 108, 1989, USA.The filters containing the lambda DNA were incubated in a hybridizationbuffer (50% formamide-1% bovine serum albumin-0.1% polyvinylpyrrolidone-0.1% ficoll-5 ×SSPE (see Molecular Cloning)-0.1% SDS-100μg/ml salmon sperm DNA) at 42° C. for 4 hours, in the same buffer butcontaining ³²P-labelled probe (ca. 200 bp, ca. 1×10⁷ cpm/ml) at 42° C.for 18 hours and in 2×SSC (see Molecular Cloning) containing 0.05% SDSat 42° C. successively to remove the excess probe. The filters wereexposed to an X-ray film HR-H (Fuji Film, Japan) at −80° C. for 18hours. As a result of the first screening of lambda phage library, about30 positive phages were obtained from 72,000 plaques.

(3) Southern blotting

One of the positive phage DNAs was digested with EcoRI and SalI (NipponGene, Japan) and subjected to 0.8% agarose gel electrophoresis. The DNAfragments separated on the gel were transferred onto a nitrocellulosefilter by electroblotting. Approximately 6 kbp DNA fragment wasidentified to hybridize the ³²P-labelled probe. It was cloned intobetween the EcoRI site and SalI site of pUC19 (Nippon Gene, Japan) togive pUC19SD5 (FIG. 2).

EXAMPLE 5 DNA sequence analysis of SDH gene

(1) Construction of the plasmids for DNA sequencing Construction ofplasmid pSD5RRV

pUC19SD5 was digested with EcoRV (Toyobo, Japan). From among the threebands separated by 1.5% agarose gel-electrophoresis, 1.1 kbp DNA whichhybridizes the ³²P-labelled probe was isolated and cloned into the SmaIsite of pUC18 to give the plasmid pSD5RRV.

(2) Construction of plasmid pSD5RVS

pUC19SD5 was digested with EcoRV and Eco47III (Toyobo, Japan). The largeDNA (ca. 5,700 bp) was isolated and self-ligated with T4 DNA ligase(Takara Shuzo, Japan) to give the plasmid pSD5RVS.

(3) DNA sequence analysis

DNA sequence analysis of the template DNA (pSD5RRV and pSD5RVS) wasperformed by dideoxy termination method with 370A DNA sequencer (AppliedBiosystems, USA) according to appended protocols. The M13 sequencingprimers, universal and reverse primers (New England Biolabs, USA) wereused for the first sequencing. Based on the DNA sequence determined bythe first sequence analysis, the following primers were synthesized andused for further DNA sequence analyses. The synthesized primers usedwere as follows (SEQ ID NO: 11-15).

Primer 1 (12 mer): 5′>CTG TGT TCT CGC<3′

Primer 2 (15 mer): 5′>TCG GTT TCG CGA AGA<3′

Primer 3 (16 mer): 5′>CGT CTT CAA CGG AAC G<3′

Primer 4 (16 mer): 5′>GGA GTG ACG TCC GTT C<3′

Primer 5 (16 mer): 51′>GAG ATG TTC TCC CAG C<3′

As a result of the analysis, an open reading frame (ORF) consisting of1596 base pairs was found. The amino acid sequence encoded by thenucleotide sequence beginning from the initiation codon (ATG) of the ORFcoincided with the amino acid sequence of SDH which was obtained inExample 2, and the theoretical molecular weight of the protein encodedby the ORF, of 58 kd coincided well with the apparent molecular weightof SDH, 58 kd by SDS-PAGE. Therefore, the ORF was determined to be theSDH gene.

The nucleotide sequence of the SDH gene is shown in the SequenceListing, Sequence No. 3 to be mentioned later, and the amino acidsequence deduced from the nucleotide sequence is shown in Sequence No.1.

EXAMPLE 6 Expression of SDH gene in E. coli

(1) Cultivation of the transformed (transfected) E. coli

A single colony of E. coli JM109 transformed with pUC19SD5 (E. coliJM109-pUC19SD5) was inoculated into 100 ml of a medium containing 1%Bactotrypton (Difco Labs., USA), 0.5% yeast extract, 0.5% sodiumchloride and 0.1% glucose (pH 7.2) in a 500 ml flask and cultivated at30° C. for 18 hours. A portion of the cultured broth (3 ml of each) wastransferred to two media containing 1% Bactotrypton, 0.5% yeast extract,0.5% sodium chloride, 1% glycerol, 0.3% KH₂PO₄, 0.8% Na₂PO₄.12H₂O (pH6.8), 1% L-sorbose and 100 μg/ml of ampicillin in a 500 ml Erlenmeyerflask. The resultant mixtures were cultivated at 25° C. for 3 days. Thecultured broth (total 200 ml) was harvested by centrifugation at 6,000rpm at 4° C. for 10 minutes. The cells were washed twice with saline,suspended in 5 ml of the same solution, and disrupted by sonication at30 second intervals for a total sonication time of 2 minutes underice-cooling. The resultant cell lysate was stored at −20° C. until usefor an enzyme assay.

(2) Assay of SDH activity

SDH expressed activity was assayed by determining the amount of thereaction product, L-sorbosone, using high performance liquidchromatography (HPLC). The reaction mixture consisting of 1% L-sorbose,1 mM phenazine methosulfate, 0.1 M phosphate buffer (pH 8.0) and thesonicated cell lysate was incubated at 30° C. with shaking for 5 hours.The reaction was stopped by adjusting pH to 2 with 6 N sulfuric acid.The reaction mixture was centrifuged at 6,000 rpm at 4° C. for 10minutes and a portion of the supernatant was directly analyzed by HPLCwith a #3011 N column (4.6 mm inner diameter×300 mm, Hitachi, Japan).The mobile phase was 1 M borate buffer (pH 9.5) containing 0.02 Mbenzamidine hydrochloride and 0.25 M potassium sulfate which were usedfor a post column labelling method, at a flow rate of 0.8 ml per minute.The post column labelling reaction was performed at 80° C. in a Tefrontube (0.5 mm inner diameter×10 m). Detection of the labelled compoundwas carried out by monitoring fluorescence (Ex. 315 nm, Em. 405 nm). Asshown in the following Table 3, the sonicated cells containing plasmidpUC19SD5 had an ability to convert L-sorbose to L-sorbosone. However, noactivity was found in the cell lysate treated at 100° C. or the cellswithout the plasmid. These results indicate that the recombinant plasmidpUC19SD5 contains the gene encoding L-sorbose dehydrogenase, whichexpressed in E. coli JM109.

TABLE 3 L-sorbosone Strain Treatment (μg/ml) JM109 (pUC19SD5) Sonication2,310 JM109 (pUC19SD5) Sonication, boiling   24 JM109 Sonication   21Basal —   33

EXAMPLE 7 Purification of SNDH from Gluconobacter oxydans T-100

(1) Preparation of crude enzyme solution

The cells (ca. 10 g, wet weight) obtained in Example 1 (2) weresuspended in 40 ml of 10 mM phosphate buffer (pH 7.0) under ice-cooling,disrupted by sonication, and centrifuged at 8,000 rpm at 4° C. for 10min. The supernatant was ultracentrifuged at 32,000 rpm at 4° C. for onehour. The resulting supernatant was used for further determinations asan SNDH crude enzyme solution.

(2) Ion-exchange chromatography

The SNDH crude enzyme solution (45 ml) was passed through QAE-TOYOPEARL550 C column (1.6 cm inner diameter×30 cm, Toso Co. Ltd., Japan)previously equilibrated with 10 mM phosphate buffer (pH 7.0). The columnwas washed with the same buffer and eluted with a linear gradient ofsodium chloride of from 0 M to 0.4 M in the same buffer. The activity ofthe enzyme was assayed by the method by SUGISAWA et al. (Agric. Biol.Chem., 55, 665-670, 1991), by measuring the amount of NADH produced bythe reaction in the presence of 13.7 μM L-sorbosone and 0.73 μM AND in50 mM phosphate buffer, which was determined by the absorbance at 340nm. The active fractions (ca. 15 ml) were pooled and diluted 5-fold withphosphate buffer for use in the purification step to follow.

(3) Blue Sepharose chromatography

The enzyme solution (ca. 75 ml) obtained in (2) was passed through BlueSepharose column (1.0 cm inner diameter×7 cm, Pharmacia, Sweden)previously equilibrated with phosphate buffer. The column was washedwith the same buffer and eluted with a linear gradient of sodiumchloride of from 0 M to 0.6 M in the same buffer. The respectivefractions were analyzed by polyacrylamide gel electrophoresis in thepresence of sodium dodecyl sulfate (SDS-PAGE) and by enzyme assay ofExample 7 (2). As a result, it was found that the protein having amolecular weight of 50 kd by SDS-PAGE corresponded to the enzymeactivity, suggesting that said 50 kd protein was the desired SNDHmolecule.

EXAMPLE 8 SNDH amino acid sequence analysis

The active fraction (75 μl) obtained in Example 7 (3) was subjected toSDS-PAGE and the separated protein was blotted on a polyvinylidenedifluoride (PVDF) membrane. The membrane containing 50 kd proteinstained by Coomassie Brilliant Blue was cut out and washed withdistilled water. The membrane piece was sequenced with an automatedprotein sequencer Model 470A (Applied Biosystems Inc., USA) forN-terminal amino acid sequence analysis. The results are shown belowwherein Xaa is an unidentified amino acid. N-terminal amino acidsequence (SEQ ID NO: 6):

Asn—Val—Val—Ser—Lys—Thr—Val—Xaa—Leu— EXAMPLE 9 DNA sequence analysis ofSDNH gene

(1) Construction of plasmid for DNA sequence analysis

The plasmid pUC19SD5 (FIG. 2) obtained in Example 4 (3) was digestedwith SalI (Nippon Gene, Japan) and EcoRV (Toyobo, Japan) and subjectedto 0.8% agarose gel electrophoresis. Of the separated DNA fragments, thefragments of about 600 bp and 4,300 bp were separated from the gel. Theformer was inserted into SmaI site of pUC18 to construct pSD6RRV. Thelatter was filled with DNA polymerase Klenow fragment (Takara Shuzo;Japan) to give a blunt-ended SalI cleavage site, followed byself-ligation to construct circular plasmid pSD5RIRV. This plasmidpSD5RIRV was digested with EcoRI and MluI, and about 3,400 bp fragmentwas isolated. Blunting and circularizing in the same manner as abovegave pSD5MRV.

(2) DNA sequence analysis

DNA sequence analyses of the template DNA (pSD5MRV, pSD6RRV and pSD5RRVused for SDH nucleotide sequence analysis) were performed by dideoxytermination method with 370A DNA sequencer (Applied Biosystems, USA).The M13 sequencing primers, universal and reverse primers (New EnglandBiolabs, USA) were used for the first sequencing. Based on the DNAsequences determined by the first sequencing, the following primers weresynthesized and used for further DNA sequence analyses. The synthesizedprimers used were as follows.

Primer 1 (15 mer); 5′>TGATGGAGAATGGCG<3′

Primer 2 (15 mer); 5′>GTAATCAGACCGACG<3′

Primer 3 (15 mer); 5′>TTCATTCTCGCATCC<3′

Primer 4 (15 mer); 5′>GATCTCACCTTTCGC<3′

Primer 5 (15 mer); 5′>CACGGATGTGAAGCC<3′

Primer 6 (15 mer); 5′>GATCCTGTGTGAGCG<3′

Primer 7 (15 mer); 5′>GCGATGTCATCACGG<3′

As the result of the above analyses, an open reading frame (ORF)consisting of 1497 bp was found in the upstream of 5′-side of SDH gene.The amino acid sequence encoded by the nucleotide sequence beginningfrom the initiation codon (ATG) of the ORF coincided with the N-terminalamino acid sequence of SNDH which was obtained in Example 8, and thetheoretical molecular weight of the protein encoded by the ORF, of 53 kdcoincided well with the molecular weight of SNDH, 50 kd by SDS-PAGE.Therefore, the ORF was considered to be the SNDH gene. The nucleotidesequence of the SNDH gene is shown in the Sequence Listing, Sequence No.4 to be mentioned later, and the amino acid sequence deduced from thenucleotide sequence is shown in Sequence No. 2.

EXAMPLE 10 Expression of SNDH gene in E. coli

(1) Cultivation of the transformed E. coli

In the same manner as in Example 6, cell lysate of E. coliJM109-pUC19SD5 was obtained and stored at −20° C. until use for anenzyme assay.

(2) Assay of SNDH activity

SNDH expressed activity was assayed by determining the amount of thereaction product, 2KLGA, using HPLC. The reaction mixture consisting of1% L-sorbosone, 0.5 mM AND, 0.1 M phosphate buffer (pH 8.0) and thesonicated cell lysate was incubated at 30° C. with shaking for 5 hours.The reaction was stopped by adjusting pH to 2 with 6 N sulfuric acid.The reaction mixture was centrifuged at 6,000 rpm at 4° C. for 10 min,and a portion of the supernatant was analyzed by HPLC (column CapcellpakNH₂; 4.6 mm inner diameter×250 mm, Shiseido, Japan). The mobile phasewas 20 mM sodium phosphate buffer (pH 3.0) containing 30% acetonitrileat a flow rate of 1.2 ml per minute. Detection was carried out bymeasuring ultraviolet absorption at 210 nm.

As a result, the mixture containing the sonicated cell lysate of E. coliJM109-pUC19SD5 transformed with the plasmid pUC19SD5 produced 5690 μg/mlof 2KLGA, demonstrating the ability to convert L-sorbosone to 2KLGA.While the host of the transformant, E. coli JM109 itself had an abilityto convert same into 2KLGA, its ability was about one-second (2170μg/ml) of the ability possessed by the transformant, thus suggestingevident enhancement of the ability of the transformant to convert sameinto 2KLGA, that is, SNDH activity. Therefrom it was made clear that therecombinant plasmid pUC19SD5 had a gene encoding SNDH, and the ORFconsisting of 1497 bp at the upstream of 5′-side of the SDH gene foundin Example 9 was an SNDH gene.

Industrial Applicability

The SDH and SNDH of the present invention are useful enzymes havingpreferable properties in the production of 2-keto-L-gulonic acid, aswell as L-ascorbic acid. According to the production method of thepresent invention, the SDH and SNDH having such preferable propertiescan be produced in large amounts by genetic engineering.

Deposit of microorganism

The cell strain used in the present invention, Gluconobacter oxydansT-100 (deposit number FERM BP-4188) has been deposited at NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Japan, since February 15, 1993.

22 530 amino acids amino acid linear peptide Gluconobacter oxydans T-100mat peptide 1..530 experimentally 1 Thr Ser Gly Phe Asp Tyr Ile Val ValGly Gly Gly Ser Ala Gly 1 5 10 15 Cys Val Leu Ala Ala Arg Leu Ser GluAsn Pro Ser Val Arg Val 20 25 30 Cys Leu Ile Glu Ala Gly Arg Arg Asp ThrHis Pro Leu Ile His 35 40 45 Met Pro Val Gly Phe Ala Lys Met Thr Thr GlyPro His Thr Trp 50 55 60 Asp Leu Leu Thr Glu Pro Gln Lys His Ala Asn AsnArg Gln Ile 65 70 75 Pro Tyr Val Gln Gly Arg Ile Leu Gly Gly Gly Ser SerIle Asn 80 85 90 Ala Glu Val Phe Thr Arg Gly His Pro Ser Asp Phe Asp ArgTrp 95 100 105 Ala Ala Glu Gly Ala Asp Gly Trp Ser Phe Arg Asp Val GlnLys 110 115 120 Tyr Phe Ile Arg Ser Glu Gly Asn Ala Val Phe Ser Gly ThrTrp 125 130 135 His Gly Thr Asn Gly Pro Leu Gly Val Ser Asn Leu Ala GluPro 140 145 150 Asn Pro Thr Ser Arg Ala Phe Val Gln Ser Cys Gln Glu MetGly 155 160 165 Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala Ser Gln Glu GlyAla 170 175 180 Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg Arg Cys SerThr 185 190 195 Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys Asn LeuThr 200 205 210 Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn GlyThr 215 220 225 Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu AsnThr 230 235 240 Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala IleGly 245 250 255 Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala AlaHis 260 265 270 Leu Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro GlyVal 275 280 285 Gly Glu Asn Leu Gln Asp His Phe Gly Val Asp Ile Val AlaGlu 290 295 300 Leu Lys Thr Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu HisTrp 305 310 315 Met Leu Trp Ala Gly Leu Glu Tyr Thr Met Phe Arg Ser GlyPro 320 325 330 Val Ala Ser Asn Val Val Glu Gly Gly Ala Phe Trp Tyr SerAsp 335 340 345 Pro Ser Ser Gly Val Pro Asp Leu Gln Phe His Phe Leu AlaGlu 350 355 360 Ala Gly Ala Glu Ala Gly Val Thr Ser Val Pro Lys Gly AlaSer 365 370 375 Gly Ile Thr Leu Asn Ser Tyr Val Leu Arg Pro Lys Ser ArgGly 380 385 390 Thr Val Arg Leu Arg Ser Ala Asp Pro Arg Val Asn Pro MetVal 395 400 405 Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp Leu Glu Thr SerAla 410 415 420 Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser Gln Pro SerLeu 425 430 435 Glu Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly Lys GlnPro 440 445 450 Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly ArgThr 455 460 465 Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp AspMet 470 475 480 Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu GlyIle 485 490 495 Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly SerAsn 500 505 510 Thr Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala AspPhe 515 520 525 Ile Gln Gly Asn Ala 530 497 amino acids amino acidlinear peptide Gluconobacter oxydans T-100 mat peptide 1..497experimentally 2 Asn Val Val Ser Lys Thr Val Ser Leu Pro Leu Lys Pro ArgGlu 1 5 10 15 Phe Gly Phe Phe Ile Asp Gly Glu Trp Arg Ala Gly Lys AspPhe 20 25 30 Phe Asp Arg Ser Ser Pro Ala His Asp Val Pro Val Thr Arg Ile35 40 45 Pro Arg Cys Thr Arg Glu Asp Leu Asp Glu Ala Val Ala Ala Ala 5055 60 Arg Arg Ala Phe Glu Asn Gly Ser Trp Ala Gly Leu Ala Ala Ala 65 7075 Asp Arg Ala Ala Val Leu Leu Lys Ala Ala Gly Leu Leu Arg Glu 80 85 90Arg Arg Asp Asp Ile Ala Tyr Trp Glu Val Leu Glu Asn Gly Lys 95 100 105Pro Ile Ser Gln Ala Lys Gly Glu Ile Asp His Cys Ile Ala Cys 110 115 120Phe Glu Met Ala Ala Gly Ala Ala Arg Met Leu His Gly Asp Thr 125 130 135Phe Asn Asn Leu Gly Glu Gly Leu Phe Gly Met Val Leu Arg Glu 140 145 150Pro Ile Gly Val Val Gly Leu Ile Thr Pro Trp Asn Phe Pro Phe 155 160 165Met Ile Leu Cys Glu Arg Ala Pro Phe Ile Leu Ala Ser Gly Cys 170 175 180Thr Leu Val Val Lys Pro Ala Glu Val Thr Ser Ala Thr Thr Leu 185 190 195Leu Leu Ala Glu Ile Leu Ala Asp Ala Gly Leu Pro Lys Gly Val 200 205 210Phe Asn Val Val Thr Gly Thr Gly Arg Thr Val Gly Gln Ala Met 215 220 225Thr Glu His Gln Asp Ile Asp Met Leu Ser Phe Thr Gly Ser Thr 230 235 240Gly Val Gly Lys Ser Cys Ile His Ala Ala Ala Asp Ser Asn Leu 245 250 255Lys Lys Leu Gly Leu Glu Leu Gly Gly Lys Asn Pro Ile Val Val 260 265 270Phe Ala Asp Ser Asn Leu Glu Asp Ala Ala Asp Ala Val Ala Phe 275 280 285Gly Ile Ser Phe Asn Thr Gly Gln Cys Cys Val Ser Ser Ser Arg 290 295 300Leu Ile Val Glu Arg Ser Val Ala Glu Lys Phe Glu Arg Leu Val 305 310 315Val Pro Lys Met Glu Lys Ile Arg Val Gly Asp Pro Phe Asp Pro 320 325 330Glu Thr Gln Ile Gly Ala Ile Thr Thr Glu Ala Gln Asn Lys Thr 335 340 345Ile Leu Asp Tyr Ile Ala Lys Gly Lys Ala Glu Gly Ala Lys Leu 350 355 360Leu Cys Gly Gly Gly Ile Val Asp Phe Gly Lys Gly Gln Tyr Ile 365 370 375Gln Pro Thr Leu Phe Thr Asp Val Lys Pro Ser Met Gly Ile Ala 380 385 390Arg Asp Glu Ile Phe Gly Pro Val Leu Ala Ser Phe His Phe Asp 395 400 405Thr Val Asp Glu Ala Ile Ala Ile Ala Asn Asp Thr Val Tyr Gly 410 415 420Leu Ala Ala Ser Val Trp Ser Lys Asp Ile Asp Lys Ala Leu Ala 425 430 435Val Thr Arg Arg Val Arg Ala Gly Arg Phe Trp Val Asn Thr Ile 440 445 450Met Ser Gly Gly Pro Glu Thr Pro Leu Gly Gly Phe Lys Gln Ser 455 460 465Gly Trp Gly Arg Glu Ala Gly Leu Tyr Gly Val Glu Glu Tyr Thr 470 475 480Gln Ile Lys Ser Val His Ile Glu Thr Gly Lys Arg Ser His Trp 485 490 495Ile Ser 1596 base pairs nucleic acid double linear DNA (genomic)Gluconobacter oxydans T-100 CDS 4..1593 experimentally 3 ATGACGAGCGGTTTTGATTA CATCGTTGTC GGTGGCGGTT CGGCTGGCTG TGTTCTCGCA 60 GCCCGCCTTTCCGAAAATCC TTCCGTCCGT GTCTGTCTCA TCGAGGCGGG CCGGCGGGAC 120 ACGCATCCCCTGATCCACAT GCCGGTCGGT TTCGCGAAGA TGACCACGGG GCCGCATACC 180 TGGGATCTTCTGACGGAGCC GCAGAAACAT GCGAACAACC GCCAGATCCC CTATGTGCAG 240 GGCCGGATTCTGGGCGGCGG ATCGTCCATC AACGCGGAAG TCTTCACGCG GGGACACCCT 300 TCCGACTTCGACCGCTGGGC GGCGGAAGGT GCGGATGGCT GGAGCTTCCG GGATGTCCAG 360 AAGTACTTCATCCGTTCCGA AGGCAATGCC GTGTTTTCGG GCACCTGGCA TGGCACGAAC 420 GGGCCGCTCGGGGTGTCCAA CCTCGCGGAG CCGAACCCGA CCAGCCGTGC CTTCGTGCAG 480 AGCTGTCAGGAAATGGGGCT GCCCTACAAC CCTGACTTCA ACGGCGCATC GCAGGAAGGC 540 GCAGGCATCTATCAGATGAC GATCCGCAAC AACCGGCGCT GCTCGACGGC TGTGGGGTAT 600 CTGCGTCCGGCTCTGGGGCG GAAGAACCTG ACGGTTGTGA CGCGGGCGCT GGTCCTGAAG 660 ATCGTCTTCAACGGAACGCG GGCGACGGGC GTGCAGTATA TCGCCAACGG CACCCTGAAT 720 ACCGCCGAAGCGAGCCAGGA AATCGTTGTG ACGGCCGGAG CGATCGGAAC GCCGAAGCTG 780 ATGATGCTGTCGGGCGTCGG GCCTGCCGCG CATCTTCGCG AAAATGGTAT CCCGGTCGTG 840 CAGGATCTGCCGGGCGTGGG CGAGAACCTT CAGGATCATT TCGGTGTGGA TATCGTAGCC 900 GAGCTCAAGACGGATGAGAG CTTCGACAAG TACCGGAAAC TGCACTGGAT GCTGTGGGCA 960 GGTCTTGAATATACCATGTT CAGATCCGGT CCCGTTGCAT CCAACGTGGT TGAGGGCGGC 1020 GCGTTCTGGTACTCGGACCC GTCATCGGGT GTTCCTGATC TCCAGTTCCA TTTTCTTGCG 1080 GAGGCTGGGGCTGAGGCTGG AGTGACGTCC GTTCCCAAGG GAGCGTCCGG GATTACGCTG 1140 AACAGCTATGTGCTGCGTCC GAAGTCTCGT GGAACTGTCC GGCTGCGTTC GGCAGATCCA 1200 AGGGTCAATCCGATGGTCGA TCCCAATTTC CTTGGAGACC CGGCCGACCT TGAGACGTCT 1260 GCGGAAGGTGTGCGCCTGAG CTACGAGATG TTCTCCCAGC CGTCTTTGGA GAAGCACATC 1320 CGGAAAACCTGTTTCTTTAG CGGTAAACAG CCGACGATGC AGATGTATCG GGACTATGCG 1380 CGGGAACATGGCCGGACGTC CTATCATCCG ACATGCACCT GCAAGATGGG TCGTGATGAC 1440 ATGTCCGTCGTCGATCCGCG TCTGAAGGTT CATGGCCTTG AGGGCATCAG GATCTGTGAC 1500 AGTTCGGTTATGCCGTCGCT GCTCGGTTCC AACACCAATG CTGCGACGAT CATGATCAGT 1560 GAGCGGGCAGCGGATTTCAT TCAGGGGAAC GCCTGA 1596 1497 base pairs nucleic acid doublelinear DNA (genomic) Gluconobacter oxydans T-100 CDS 4..1494experimentally 4 ATGAATGTTG TCTCAAAGAC TGTATCTTTA CCGTTAAAGC CGCGTGAGTTCGGATTCTTT 60 ATTGATGGAG AATGGCGCGC AGGTAAGGAT TTCTTCGATC GTTCCTCGCCGGCTCATGAT 120 GTTCCCGTCA CCCGTATTCC ACGCTGCACC CGTGAAGACC TTGATGAGGCAGTCGCTGCT 180 GCACGTCGTG CTTTCGAGAA CGGAAGCTGG GCGGGTCTGG CAGCCGCGGATCGTGCGGCG 240 GTTCTTCTGA AAGCCGCGGG CCTTCTGCGC GAGCGCCGTG ATGACATCGCTTACTGGGAA 300 GTTCTCGAAA ACGGGAAGCC CATCAGCCAG GCGAAAGGTG AGATCGATCACTGTATCGCC 360 TGTTTCGAGA TGGCGGCCGG CGCTGCGCGG ATGCTGCATG GTGATACGTTCAACAATCTG 420 GGCGAGGGGC TGTTTGGCAT GGTCCTGCGG GAGCCCATCG GTGTCGTCGGTCTGATTACG 480 CCGTGGAACT TCCCGTTCAT GATCCTGTGT GAGCGGGCGC CTTTCATTCTCGCATCCGGC 540 TGCACGCTGG TCGTCAAGCC TGCCGAAGTC ACGAGTGCCA CGACCCTTCTTCTGGCAGAA 600 ATCCTTGCCG ATGCCGGGCT GCCGAAGGGT GTCTTCAATG TCGTGACAGGCACGGGGCGC 660 ACGGTCGGTC AGGCCATGAC CGAGCATCAG GATATCGACA TGCTGTCCTTCACGGGCTCC 720 ACGGGCGTCG GCAAGTCCTG TATCCACGCG GCGGCTGACA GCAACCTGAAGAAACTTGGC 780 CTCGAACTGG GCGGCAAGAA CCCGATTGTC GTGTTCGCTG ACAGCAACCTTGAGGATGCG 840 GCCGACGCGG TAGCCTTCGG GATCAGCTTT AATACCGGGC AGTGCTGTGTGTCGTCGAGC 900 CGCCTGATCG TAGAGCGGTC CGTGGCGGAG AAGTTCGAGC GCCTCGTCGTGCCAAAAATG 960 GAGAAGATCC GCGTTGGTGA TCCGTTTGAT CCCGAAACGC AGATTGGCGCCATCACGACG 1020 GAAGCGCAGA ACAAGACCAT TCTGGACTAT ATCGCGAAAG GCAAGGCCGAGGGCGCCAAG 1080 CTGCTCTGTG GTGGCGGGAT CGTCGATTTC GGCAAGGGAC AGTATATCCAGCCCACGCTT 1140 TTCACGGATG TGAAGCCCTC GATGGGCATC GCGCGTGACG AGATTTTTGGGCCGGTTCTG 1200 GCGTCCTTCC ACTTCGATAC CGTCGATGAG GCGATCGCGA TTGCCAATGACACGGTTTAC 1260 GGCTTGGCCG CATCGGTCTG GAGCAAGGAT ATCGACAAGG CGCTTGCCGTGACCCGTCGT 1320 GTTCGTGCCG GCCGCTTCTG GGTGAACACC ATCATGAGCG GTGGTCCCGAGACGCCGCTG 1380 GGTGGTTTCA AGCAGTCGGG CTGGGGCCGT GAGGCCGGTC TGTACGGCGTTGAGGAATAT 1440 ACGCAGATCA AATCTGTCCA TATCGAAACT GGCAAACGTT CGCACTGGATTTCGTAA 1497 14 amino acids amino acid linear peptide unknown 5 Thr SerGly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala 1 5 10 9 amino acidsamino acid linear peptide unknown 6 Asn Val Val Ser Lys Thr Val Xaa Leu1 5 16 amino acids amino acid linear peptide unknown 7 Met Thr Thr GlyPro His Thr Trp Asp Leu Leu Thr Glu Pro Gln Lys 1 5 10 15 10 amino acidsamino acid linear peptide unknown 8 Leu Met Met Leu Ser Gly Val Gly ProAla 1 5 10 23 base pairs nucleic acid single linear other nucleic acid(synthetic DNA) unknown 9 ACCWSCGGCT TYGAYTAYAT HGT 23 17 base pairsnucleic acid single linear other nucleic acid (synthetic DNA) unknown 10TCCCANGTRT GNGGNCC 17 12 base pairs nucleic acid single linear othernucleic acid (synthetic DNA) unknown 11 CTGTGTTCTC GC 12 15 base pairsnucleic acid single linear other nucleic acid (synthetic DNA) unknown 12TCGGTTTCGC GAAGA 15 16 base pairs nucleic acid single linear othernucleic acid (synthetic DNA) unknown 13 CGTCTTCAAC GGAACG 16 16 basepairs nucleic acid single linear other nucleic acid (synthetic DNA)unknown 14 GGAGTGACGT CCGTTC 16 16 base pairs nucleic acid single linearother nucleic acid (synthetic DNA) unknown 15 GAGATGTTCT CCCAGC 16 15base pairs nucleic acid single linear other nucleic acid (synthetic DNA)unknown 16 TGATGGAGAA TGGCG 15 15 base pairs nucleic acid single linearother nucleic acid (synthetic DNA) unknown 17 GTAATCAGAC CGACG 15 15base pairs nucleic acid single linear other nucleic acid (synthetic DNA)unknown 18 TTCATTCTCG CATCC 15 15 base pairs nucleic acid single linearother nucleic acid (synthetic DNA) unknown 19 GATCTCACCT TTCGC 15 15base pairs nucleic acid single linear other nucleic acid (synthetic DNA)unknown 20 CACGGATGTG AAGCC 15 15 base pairs nucleic acid single linearother nucleic acid (synthetic DNA) unknown 21 GATCCTGTGT GAGCG 15 15base pairs nucleic acid single linear other nucleic acid (synthetic DNA)unknown 22 GCGATGTCAT CACGG 15

What is claimed is:
 1. A recombinant L-sorbosone dehydrogenase having anamino acid sequence of SEQ ID NO:
 2. 2. An L-sorbosone dehydrogenaseproduced by a process comprising: culturing a host cell transformed byan expression vector containing a DNA encoding the L-sorbosonedehydrogenase having the amino acid sequence of SEQ ID NO: 2 in aculture medium; and isolating said L-sorbosone dehydrogenase from theculture medium.